A noncoding RNA gene on chromosome 10p15.3 may function upstream of hTERT

Abstract

Background: We attempted to clone candidate genes on 10p 14-15 which may regulate hTERT expression, through exon trapping using 3 BAC clones covering the region. After obtaining 20 exons, we examined the function of RGM249 (RGM: RNA gene for miRNAs) we cloned from primary cultured human hepatocytes and hepatoma cell lines. We confirmed approximately 20 bp products digested by Dicer, and investigated the function of this cloned gene and its involvement in hTERT expression by transfecting the hepatoma cell lines with full-length dsRNA, gene-specific designed siRNA, and shRNA-generating plasmid.

Results: RGM249 showed cancer-dominant intense expression similar to hTERT in cancer cell lines, whereas very weak expression was evident in human primary hepatocytes without telomerase activity. This gene was predicted to be a noncoding precursor RNA gene. Interestingly, RGM249 dsRNA, siRNA, and shRNA inhibited more than 80% of hTERT mRNA expression. In contrast, primary cultured cells overexpressing the gene showed no significant change in hTERT mRNA expression; the overexpression of the gene strongly suppressed hTERT mRNA in poorly differentiated cells.

Conclusion: These findings indicate that RGM249 might be a microRNA precursor gene involved in the differentiation and function upstream of hTERT.

Figure 1

Schematic diagram showing the chromosomal location (10p15.3) of exons including RGM249. (a) Mapping of exons trapped between 10p15.1 and 10p15.3. Reference was used as an assembly database. This region does not contain any known genes except GATA3. Solid arrows indicate both ends of the sequences which BAC clones cover and show that 9447200 links to 9447201 in NT077569.2. (b) Schematic diagram showing the chromosomal location (10p15.3) of RGM249, which consists of 2 exons, genomic fragments (AL355591.3 and AL138774.5), and a genomic marker (D10S1728) containing the gene and BAC clones (H-11, M-6, and J-21) used for the mapping. The full length of the gene is 249 bp. Solid and dashed lines represent the exons trapped and their neighboring gene (GATA3), respectively.

Figure 2

A quantitative estimation in normal cells, cancer cells, and tissues. To quantify RGM249 mRNA expression in hepatocytes, hepatoma cells, and liver tissues, the copy number in 10 ng of total RNA was evaluated. (a) One-step real-time PCR was performed based on the linear correlation (P > 0.990) of the RNA controls. (b) RGM249 mRNA expression was examined in human representative organs using one-step real-time RT-PCR. The vertical line shows the copy number per 50 ng/μl total RNA (c) RGM249 mRNA expression in liver-related cells (from the left: adult liver tissues, fetal liver tissues, primary cultured hepatocytes [T], cytoplasm segment of primary hepatocytes [C], nuclear segment of primary hepatocytes [N], HLF cells, HMc-Li7 cells, HepG2 cells, and Alexander cells). (d) RGM249 mRNA expression in surgically resected liver tissues containing 33 pairs of hepatomas and adjacent noncancerous tissue. C: cancerous lesion, NC: adjacent noncancerous lesion. Cancerous lesions showed a significant upregulation of RGM249 mRNA, compared with noncancerous lesions, as determined by the t-test (P = 0.007) and paired t-test (P = 0.040). Box represents 95% confidence intervals.

Figure 3

Outlines of cloned RGM249, a possible miRNA precursor or telomerase regulated gene. (a) RGM249 sequence is shown, and the sequences used for the siRNA and shRNA designs are underlined. (b) Predictive secondary structure of RGM249 was drawn by Vienna RNA Secondary prediction and comparison . Bold line corresponds to the sequence used as the most functional siRNA or shRNA. The underlined parts show the sense and antisense sequences generating shRNA. Star (⋆) shows deletion of T or C in mt-1RGM249 or mt-2RGM249, respectively. (c) From the digestion by RNase III (Dicer), RGM249 mRNA generates 3 products ranging from 17 to 23 bp, suggesting that this gene may function as a noncoding precursor RNA gene which produces miRNAs.

Figure 4

Expression of telomerase-related genes in RNAi methods. (a) Inhibitory effect of full-length RGM249 dsRNA (100 nM dsRNA) on the transcriptional expression of telomerase-related genes in a hepatocellular carcinoma cell line (HMc-Li7) as determined by RT-PCR. RGM249, hTERT, and RPL22 were significantly suppressed. The lower blotting demonstrates gene expression using a transfection reagent without dsRNA. (b) RT-PCR showing the inhibitory effects of RGM249-specific siRNA on RGM249 mRNA, hTERT mRNA, and RPL22 mRNA in HLF cells, which express RGM249 mRNA in 4 hepatoma cell lines in the strongest manner. siRNA ① and siRNA ② correspond to those in Table 2. (c) Quantitative evaluation of the suppressive effect of siRNA in which the measurement, in the case without siRNA, was regarded as 100% for standardization (N = 5), suggesting that siRNA ① has a functional sequence. RGM249 siRNA transcriptionally suppressed more than 80% of hTERT expression and approximately 50% of RPL22 expression. RGM249 mRNA, ▯ hTERT mRNA, RPL22 mRNA. d) Quantitative evaluation of the inhibitory effect of RGM249 shRNA on RGM249 mRNA, hTERT mRNA, and RPL22 mRNA. From left, transfection using only transfection reagents, LacZ shRNA as the gene control, wtRGM249 shRNA, and mt-1RGM249 or mt-2RGM249 shRNA with a different mutation (a nucleotide T or C) in the functional sequence. A total of 5 transfections were performed and data were statistically analyzed using the Mann-Whitney test. Compared with LacZ shRNA, RGM249 and mt-1RGM249 shRNA significantly suppressed hTERT mRNA (P = 0.002 and P = 0.034, respectively). mt-1RGM249 and mt-2RGM249 correspond to those in Table 2. ▯ RGM249 mRNA, hTERT mRNA, RPL22 mRNA, *: P < 0.05; **: P < 0.01. Quantitative evaluation of the suppressive effect of shRNA in which the measurement, in the case without siRNA, was regarded as 100% for standardization (N = 5). (e) Telomerase activity was quantitatively compared using image analyzer between transfectants with LacZ shRNA (in proliferative state) and RGM249 shRNA (in senesced state) and RGM249 shRNA, and was compared with the parental cells (112 and 54, respectively, which had an intensity of 100). Although the results depended on the timing of cell harvests after transfection, telomerase activity in transfectants with RGM249 shRNA was reduced to approximately half compared with that in parental cells, and we observed a periodicity of 6 bp due to reduced telomerase in the shRNA lane. NC: telomerase negative control.

Figure 5

FACS analysis and phenotypes in transfectants by RNAi methods. (a) FACS analysis showing induction of more than half of the transfected populations into the preG1 phase of the cell cycle. As shown in the upper two parts, the increase in apoptotic cells in transfectants by RGM249 is shown as red circled areas. DNA content analysis revealed in the lower parts, that early cells showed typical cell proliferation profile, cells later in production showed reduced cell proliferation, and later cells also showed early signs of cell death (sub-G1), in both transfectants. In the bottom parts, the histogram with gating and the threshold were set properly for correctly stained cells in a clean machine. The large pink peak represents the green light signal from cells. The line segment flanking the peak is the gating band for cells. (b) Changes in phenotypes of HLF cells receiving LacZ shRNA or RGM249 shRNA are shown. The upper, middle, and bottom part shows the phenotype of transfectants, the result of β-Gal staining as an indicator of senescence, and EGFP expression as evidence of successful transfection, respectively. At 2–3 weeks after transfection, the transfectants were induced to undergo apoptosis (upper and bottom parts) through senescence (middle part) in the presence of both a selectable marker (neo) and RGM249 (data not shown).

Figure 6

A representative gene expression in microarray analysis. Representatives of regulated genes using microarray analysis performed between transfectants with LacZ shRNA and RGM249 shRNA are shown as a gene symbol (description) and ×fold. Following transfection of the RNA generating-vector and presumably the generation of RNA molecules, many transcriptional factors, the molecules involved in RNA biogenesis, and tumor-related genes altered the expression level.

Figure 7

A representative miRNA expression in microRNAarray analysis and predictive target genes. (a) Representative genes predicted to be commonly targeted by RGM249 miRNAs or siRNAs are shown. 4 miRNAs were predicted from the RGM249 sequence by miRBase or miRNAMAP and it was predicted that the miRNAs might inhibit the translation of genes involved in oxidation and RAS activation. As reported previously in association with hTERT expression, PRKCA, MAPK1, and AKT1 were included in the targeted genes. (b) Seven miRNAs upregulated more than twofold in RGM249 shRNA-transfectants by microRNAarray analysis performed between transfectants with LacZ shRNA and RGM249 shRNA are shown. Some of these 7 miRNAs have been indicated in the oncogenesis of oral squamous cell carcinoma (unpublished data). (c) Representative genes targeted by miRNAs which changed more than twofold when induced by shRGM249RNA are shown. The genes were predicted mainly using miRNAMAP. Three miRNAs were presumably targeted towards miRNA processing proteins, transcriptional factors, telomere-related genes, TERT-related genes, tumor suppressor-related genes, and cell cycle-related genes.